Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Apr 22;106(2):265-276.e6.
doi: 10.1016/j.neuron.2020.01.037. Epub 2020 Feb 27.

Activity-Dependent Plasticity of Axo-axonic Synapses at the Axon Initial Segment

Alejandro Pan-Vazquez  1 , Winnie Wefelmeyer  1 , Victoria Gonzalez Sabater  1 , Guilherme Neves  2 , Juan Burrone  3
Affiliations

Activity-Dependent Plasticity of Axo-axonic Synapses at the Axon Initial Segment

Alejandro Pan-Vazquez et al. Neuron. .

Abstract

The activity-dependent rules that govern the wiring of GABAergic interneurons are not well understood. Chandelier cells (ChCs) are a type of GABAergic interneuron that control pyramidal cell output through axo-axonic synapses that target the axon initial segment. In vivo imaging of ChCs during development uncovered a narrow window (P12-P18) over which axons arborized and formed connections. We found that increases in the activity of either pyramidal cells or individual ChCs during this temporal window result in a reversible decrease in axo-axonic connections. Voltage imaging of GABAergic transmission at the axon initial segment (AIS) showed that axo-axonic synapses were depolarizing during this period. Identical manipulations of network activity in older mice (P40-P46), when ChC synapses are inhibitory, resulted instead in an increase in axo-axonic synapses. We propose that the direction of ChC synaptic plasticity follows homeostatic rules that depend on the polarity of axo-axonic synapses.

Keywords: Chandelier; GABA; activity-dependent; axo-axonic; axon initial segment; development; homeostatic; interneuron; plasticity.

PubMed Disclaimer

Conflict of interest statement

Declaration of Interests The authors declare no competing interests.

Figures

Figure 1
Figure 1
Development of Chandelier Cells and Axo-axonic Synapses in Somatosensory Cortex (A) Genetic strategy and timeline for tamoxifen injection for labeling ChCs in Nkx2.1-CreER+/−;Ai9 mice, cranial window implantation, and repeated in vivo imaging. (B) In vivo image (P16) and reconstructions (P12–P16) of a ChC. Scale bar, 40 μm. (C) Number of cartridges for individual ChCs during development (gray) and mean cartridge number (black, n = 4 ChCs, 3 mice). (D) Image of a ChC (red) and AISs (green) at P18. Connection probability was defined as the percentage of AISs with ChC overlap within a 90 μm radius (white circle). (E) Average connection probability of ChCs across development (4–5 ChCs, 2–4 mice per time point), with a sigmoidal fit (red). (F) Images of axo-axonic synapses located on an AIS and expressing VGAT at P14 and P16. Scale bar, 2 μm. (G–I) Average number (G) and density (H) of axo-axonic boutons as well as (I) AIS length across development, from fixed tissue samples (n = 26–81 AISs, 2–4 mice per time point). The green shaded area highlights the period of rapid synaptic development. (J) ChCs expressing ChR2 were stimulated with light and GABAergic PSCs recorded in nearby pyramidal cells (left). Example responses (middle) and average GABAergic PSC amplitude (right) in immature and mature networks (∗∗p < 0.01, Mann-Whitney test. n = 10–11 neurons, 4 mice, per condition). Plots show mean ± SEM. See also Figure S1.
Figure 2
Figure 2
Activity-Dependent Plasticity of Axo-axonic Synapses (A) Schematic of DREADD receptor hM3Dq (top left), timeline of CNO application (below), and logic of experimental design resulting in ChCs contacting hM3Dq+ (green) and hM3Dq– (gray) pyramidal cells in the same network. (B) Connection probability of ChCs at P18 in a 90 μm radius (white circle) following injection of CNO into control mice (CNO control), into hM3Dq expressing mice (hM3Dq network) or saline into hM3Dq expressing mice (saline control) (chi-square test. n = 270–390 cells, 3–4 mice, per condition). (C) Example images showing axo-axonic boutons overlapping with the AIS at P18. (D and E) Cumulative distribution of and average axo-axonic bouton density (D) and number (E) (one-way Kruskal-Wallis with Dunn’s multiple comparison test. n = 20–132 cells, 3–4 mice, per condition). (F) Example images of axo-axonic cartridges. (G) Cumulative distribution and average cartridge size (Kruskal-Wallis with Dunn’s multiple comparison test, n = 21–40 neurons, 3–4 mice, per condition). (H and I) Schematic diagram of patch-clamp experiment (H) and representative GABAergic PSCs (I) from pyramidal cells after optogenetic ChC stimulation for CNO control (blue) and hM3Dq-network (black) conditions. (J and K) Average GABAergic PSC amplitudes (J) and average failure rates (K) (chi-square test, n = 13–14 neurons, 4 mice, per condition). (L) Connection probability (chi-square test, n = 36–57 neurons, 5 mice, per condition). ∗p < 0.05 ∗∗∗p < 0.001. Bar plots show mean ± SEM. See also Figures S2, S3, and S5.
Figure 3
Figure 3
Axo-axonic Plasticity Matches GABAergic Polarity (A) Schematic showing iontophoresis experiment. A pipette containing GABA was placed near the AIS of a pyramidal neuron expressing Ace2N-mNeon. (B) Example traces obtained from imaging somatic responses to iontophoretic GABA application (orange area) at the AIS of two different cells. Left, a depolarizing response that is blocked by the application of 10 μM SR95531 (GABAzine). Right, example trace showing a hyperpolarizing response. (C) Classification of responses into hyperpolarizing (blue), depolarizing (red), and putative shunting (black) for all cells tested. (D) Schematic showing experimental logic: ChCs were patch-clamped to evoke APs, and responses were imaged from the soma of nearby pyramidal cells expressing Ace2N-mNeon. (E) Left, example image of Ace2N-mNeon cells, with somas outlined in different colors. Right, change in fluorescence of corresponding cells following ChC stimulation (top; 5 APs, 50 Hz) (∗denotes time-locked event 3x >baseline standard deviation in cell 3). Vertical lines denote start and end of expected response period, allowing up to 50 ms for decay of GABAergic responses (Figure 2I). (F) Heatmap showing Z scores of fluorescence responses following ChC stimulation for all 22 pyramidal cells tested. Mean Z score across all cells is shown below. See also Figures S4 and S6.
Figure 4
Figure 4
Axo-axonic Plasticity Is Reversible (A) Logic of experimental design and timeline of CNO application, including the recovery period. (B) Example image of a ChC and connection probabilities within a 90 μm radius at P22, after the recovery period (chi-square test, n = 180–390 AISs from 2–3 mice, per condition). (C) Example images of axo-axonic synapses and AISs following the recovery period. (D and E) Cumulative distribution of and average axo-axonic bouton density (D) and number (E) (#p < 0.05, Kruskal-Wallis test with Dunn’s post hoc comparisons; ∗∗∗p < 0.001 for P18 versus P22 hM3Dq-network, Mann-Whitney test. n = 41–89 neurons, from 2–3 mice, per condition). Bar plots show mean ± SEM. See also Figure S5.
Figure 5
Figure 5
Axo-axonic Plasticity Is Cell Autonomous (A) Timeline of CNO delivery (top left) and strategy for viral delivery of GFP and hM3Dq to ChCs (bottom left). Experimental conditions of viral strategy (right). For CNO control, those ChCs infected with GFP virus only were analyzed, whereas, for the hM3Dq+ condition, ChCs expressing both GFP and hM3Dq-mCherry were analyzed. (B) Example images of P18 control and hM3Dq+ ChCs. (C) Connection probability within a 90 μm radius (white circle in B) for control and hM3Dq+ ChCs. chi-square test, n = 120–150 AISs per condition, from 3 mice. (D) Example images of P18 control and hM3Dq+ axo-axonic boutons. (E and F) Cumulative distribution of and average axo-axonic bouton density (E) and number (F). ∗p < 0.01 Mann-Whitney test, n = 60 AISs per condition, from 3 mice. Bar plots show mean ± SEM. See also Figure S5.
Figure 6
Figure 6
Axo-axonic Plasticity Matches Developmental Changes in GABAergic Polarity (A) Left, schematic and example image of iontophoresis experiment. A pipette containing GABA was placed near the soma (pipette in red) of a pyramidal neuron expressing Ace2N-mNeon (cyan). Right, example hyperpolarizing voltage response obtained from imaging somatic responses to iontophoretic GABA application (orange area) at the soma of a P46 pyramidal cell. (B) Classification of responses into hyperpolarizing (blue), depolarizing (red), and putative shunting (black) for all cells tested. (C) Timeline for CNO delivery in adult mice. (D) Example images of axo-axonic boutons in the different conditions at P46. (E and F) Cumulative distribution and average axo-axonic bouton number (E) and density (F) (# denotes p < 0.05 for one-way ANOVA without reaching significance in post hoc comparisons; ∗p < 0.05 denotes Kruskal-Wallis and Dunn’s post hoc comparisons test, n = 44–72 neurons per condition, 3–5 mice, per condition). (G and H) Example image (G) and connection probabilities (H) within a 90 μm radius (white circle) at P46 (chi-square test, n = 150–180 AISs, 3–4 mice, per condition). Bar plots show mean ± SEM. See also Figures S4–S6.
See this image and copyright information in PMC

Comment in

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Andreae L.C., Burrone J. The role of neuronal activity and transmitter release on synapse formation. Curr. Opin. Neurobiol. 2014;27:47–52. - PMC - PubMed
    1. Baalman K.L., Cotton R.J., Rasband S.N., Rasband M.N. Blast wave exposure impairs memory and decreases axon initial segment length. J. Neurotrauma. 2013;30:741–751. - PMC - PubMed
    1. Ben-Ari Y. Excitatory actions of gaba during development: the nature of the nurture. Nat. Rev. Neurosci. 2002;3:728–739. - PubMed
    1. Ben-Ari Y., Gaiarsa J.L., Tyzio R., Khazipov R. GABA: a pioneer transmitter that excites immature neurons and generates primitive oscillations. Physiol. Rev. 2007;87:1215–1284. - PubMed
    1. Bonifazi P., Goldin M., Picardo M.A., Jorquera I., Cattani A., Bianconi G., Represa A., Ben-Ari Y., Cossart R. GABAergic hub neurons orchestrate synchrony in developing hippocampal networks. Science. 2009;326:1419–1424. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources